Genomics Core: Illumina GoldenGate SNP Genotyping Assay

1. Illumina BeadArray Technology
2. Illumina Golden Gate SNP Genotyping Arrays (Sentrix BeadChip and Array Matrix)
3. DNA Isolation Protocols
4. Services Offered, Sample Submission / Data Output Files
5. Quality Control
6. Pricing
7. Core Contact Information

Remember: High quality DNA equals successful genotyping.

The Genomics Core is equipped with an Illumina Beadstation and employs experienced, well-trained personnel to handle Illumina BeadChip and Array Matrix based genotyping projects.  However, successful genotyping studies start before DNA samples are submitted to the Core: at the desk where the experiment is planned and at the bench where the DNA sample is prepared and its purity and concentration are determined: high quality DNA samples are necessary to obtain reliable genotyping results.   Therefore, investigators preparing for genotyping experiments need to do the following:

• Schedule an orientation session with the Core manager ( faberp@ccf.org ), then;
• Prepare high quality DNA.
• Determine concentration of DNA samples (PicoGreen® assay is recommended).
• Normalize DNA samples to 50 ng/uL in TE 8.0 (10mM Tris-HCl pH 8.0, 1mM EDTA).
• Submit DNA samples in a standard, clearly labelled 96-well plate. Provide at least 20 uL normalized DNA solution for each sample. Again: DNA must be in 1 x TE 8.0!!

1) Illumina BeadArray Technology

Illumina arrays are based on the ordered arraying of oligo-coated beads. Each oligonucleotide (bead type) represents a a specific SNP locus. Since each bead type is present an average of 30 times on each array, each Goldengate genotyping call is thus the result of the mean intensity of all 30 replicates, resulting in industry-leading accuracy, reproducibility, and call rates (all >99 %).

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2) Illumina Golden Gate SNP Genotyping Arrays (Sentrix BeadChip and Array Matrix)

Illumina offers two different array platforms for SNP genotyping: the Sentrix® Array Matrix (96 samples) and the Sentrix® BeadChip (16 samples / chip).

Available Illumina Goldengate SNP panels:
Several fixed-content SNP panels are available for linkage analysis in both human and mouse, fine mapping in the MHC region and association mapping in disease-specific candidate genes.

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3) DNA isolation protocols

While the protocols described below represent by no means the only possible methods to isolate the high quality/purity DNA necessary for array analysis they do represent two protocols that clients of the Genomics Core have successfully used.

DNA origin: Mouse tail snips

1. Proteinase K based digestion of tail tissue (contact Core for precise protocol).
2. QiaAmp DNA microkit (catalog 56304)

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4) Services Offered, Sample Submission / Data Output Files

A) Standard Goldengate Service:

Clients will:

• Perform DNA isolation and bring high quality DNA (50 ng/ul) to the Genomics Core

Genomics Core personnel will:

• Perform the GoldenGate Assay protocol for DNA samples
• Perform BeadChip/Array Matrix hybridization and washing.
• Perform BeadChip scan and perform genotype calling using Illumina’s Beadstudio software to asses the quality of the experiment.

Clients will:

• Perform subsequent data analysis using either the Illumina software (available on a Core computer) or other means.

B) Sample Submission:

Purchase of Illumina BeadChip/Array Matrix & Associated Reagents:

Clients are responsible for purchasing the Illumina BeadChip/Array Matrix and associated reagents.  The company contact info is: llumina East, 50 Barnes Park North Wallingford, CT 06492, USA Phone: 203-678-2400 Fax: 203-265-9109 Email: info@illumina.com

We ask that you have the BeadChip/Array Matrix and associated reagents delivered to Dr. Pieter Faber in the Genomics Core, NE5-253. After receiving the Illumina reagents we will inspect the items and properly store them until use.

Illumina Whole Genome Gene Expression Request form.

Every client must fill out an Illumina Golden Gate Assay Request Form and bring this to the Core when submitting DNA sample(s). Per sample 20ul of the DNA is needed which must be provided at a concentration of 50 ng/ul. DNA samples, accompanied with the request form, should be delivered in person to either Dr. Pieter Faber at NE5-253 where they will be stored at -4°C.  Projects will be handled in the order received.  At the time of submission a time-frame for finishing the project will be given   which will depend on the size of the project and other ongoing Illumina projects.

B) Data Output Files:

The full service data output file will contain the raw image data files (*.idat, *.jpeg, and *.xml) generated after scanning the BeadChip/Array Matrix.

Note: Illumina BeadStudio software can be used to analyze the Golden Gate data for genotyping scores and will be available through the Genomics Core to all Clients.

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5) Quality Control

Illumina BeadChip experiments come with built in controls that measure allele-specific extension, PCR uniformity, extension gap, gender, first hybridization controls, second hybridization controls, and contamination detection controls. These control oligos (with the exception of second hybridization controls) are designed to human genomic DNA sequences.

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6) Pricing

7) Core contact Information

• Pieter Faber, PhD, Manager.

The Genomics Core is located in the NE-building (NE5-253; phone: 216-444-0974). Core e-mail: faberp@ccf.org

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